C-P-015 Development of antioxidant soluble drinking powder from mamao (Antidesma ghaesembilla) fruit extract Pongtip Sithisarn* a, Wandee Gritsanapan a a Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand 10400 *pongtip.sit@mahidol.ac.th phone +66-26448677 ext. 5521 fax +66-26448701 ABSTRACT Antidesma ghaesembilla or mamao is a plant in Euphorbiaceae family. This plant has reddish purple fruits which are edible and become more popular in the recent years as beverages and health products. This experiment was set up in order to investigate free radical scavenging activities of extracts from the fruits of mamao prepared by different methods of extraction and drying including decoction, fresh squeezing and maceration using 1,1-diphenyl- 2-picrylhydrazyl (DPPH) assay. Folin-Ciocalteu and ph differential methods were also conducted to quantitatively analyze of total phenolic and total anthocyanin contents of the extracts. Finally, the extract from the most suitable extraction and drying methods was selected for development of health supplement product. Decoction and drying by evaporation on a water bath promoted extract of mamao ripe fruits with the strongest free radical scavenging activity (IC 50 = 72.42 ± 3.52 µg/ml) with high amount of total phenolic and total anthocyanin contents of 1.22 ± 0.36 g gallic acid equivalent in 100 g extract (g%gae) and 7.09 ± 0.24 g cyanidin-3-glucoside equivalent in 100 g extract (g%c- 3-GE), respectively. This extract was developed as soluble drinking powder by wet granulation method and qualitatively controlled by evaluations of loss on drying, thin layer chromatographic (TLC) and infrared spectroscopic (IR) fingerprints. The obtained product is pinkish red powder that contained 3.08 ± 0.81 g%c-3-ge in 1 sachet (14 g) and exhibited free radical scavenging activity equivalent to 0.004 g of vitamin C. The information from this study could be used as guideline for the developments of antioxidant products from mamao fruit extracts in the future. Keywords: Antidesma ghaesembilla, Mamao, Free radical scavenging activity, Total phenolic, Total anthocyanin, Soluble drinking powder www.natpro5.psu.ac.th 278
1. INTRODUCTION Many degenerative pathologies and diseases such as cardiovascular disease, Alzheimer s, atherosclerosis, inflammation and cancer are considered to relate to the excess production of free radicals [1]. To balance the obtaining and eliminating of free radicals in body system, consumption of antioxidant diets including fruits and vegetables could be the solutions to control oxidative stress in human being. Antidesma ghaesembilla which is called in Thai as mamao is a plant in Euphobiaceae family. This plant is a tree up to 20 m tall with simple alternate leaves and multiple fruits [2]. The leaves and the fruits have been traditionally used to treat anemia and promote blood circulation while the barks have been used as astringent and tonic [2]. Nowadays, the ripe fruits of mamao which have red to dark purple color have been developed as various nutritional products such as wine, jam, and fruit juice [3]. Since there are various nutritional applications from mamao fruits, however, there has been no report concerning the biological activities and phytochemicals in this plant. Therefore, this study was set up in order to investigate for antioxidant activity of extracts from the fruits of mamao using DPPH scavenging assay and quantitatively analyze for active components including phenolics and anthocyanins contents by Folin-Ciocalteu and ph differential methods, respectively. Moreover, investigation of suitable fruit extract preparations such as extraction and drying methods and product development were also conducted. 2. MATERIALS AND METHODS Plant materials Purple and red fruits of mamao (A. ghaesembilla) were collected from Nong Khai province, Thailand in January, 2012. Plant materials were identified by Assoc. Prof. Dr. Wandee Gritsanapan, Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand. The samples were cleaned and air dried then the seeds were removed and the flesh was cut into small pieces. The flesh of purple and red fruits of mamao was separately extracted using the extraction procedures below. Decoction The flesh of purple fruits of mamao was boiled (80 C) with distilled water (plant/water ratio 1:10 w/v) for 8 h, then filtered. The filtrate was dried using spray drying (130 C), freeze drying (-45 C) and evaporation on a water bath method (60 C) to obtain dried purple fruit decoction extracts of spray drying, freeze drying and evaporation (PDS, PDF and PDE, respectively). The red fruits of mamao were also extracted by decoction using the same decoction protocol then freeze drying to obtain red fruit decoction freeze drying extract (RDF). Maceration The flesh of purple fruits of mamao was separately macerated with 95% ethanol, methanol and acetone (plant/water ratio 1:10 w/v) for 48 h with occasional shaking then filtered. The filtrate was evaporated under reduced pressure to dryness. These dried purple fruit ethanol, methanol and acetone extracts were labeled as PME, PMM and PMA, respectively. Squeezing The flesh of purple fruits of mamao was minced with distilled water (plant/water ratio 1:10 w/v) using electronic blender then filtered. The filtrate was dried using freeze drying method to yield purple fruit squeezing extracts (PS). All extracts were analyzed for antioxidant activities, total phenolic and total anthocyanin contents using DPPH scavenging assay, Folin-Ciocalteu method and ph differential method, respectively. The extract that promoted the best quality was selected for further product development. Determination of free radical scavenging activity using DPPH scavenging assay The free radical scavenging effect of all mamao fruit extracts as well of standard trolox corresponding to the quenching ability to DPPH radical was carried out as described by Yamasaki et al. [4]. DPPH solution was added to the sample solution in the same volume. The absorbance at 517 nm was determined after 15 minutes of the reaction and percentage of scavenging and IC 50 were calculated. Each sample was assayed in triplicate and the average of IC 50 value was calculated. Determination of total phenolic content using Folin-Ciocalteu method Using the method applied from Naithani et al. [5], plant extract solutions were oxidized with Folin-Ciocalteu reagent and the reactions were neutralized by sodium carbonate solution. The absorbance of the resulting blue colored solution was measured at 765 nm after 120 min. Total phenolic content was expressed as g gall acid equivalent in 100 g extract (g% GAE). Each sample was done in triplicate and the average result was calculated. www.natpro5.psu.ac.th 279
Determination of total anthocyanin content using ph differential method Applied the methods from Sutharut and Sudarat [6], plant extract solution were separately prepared in potassium chloride buffer, ph 1.0 and sodium acetate buffer, ph 4.5 and stayed in room temperature for 15 minutes. Then the absorbance of each solution was measured at 510 and 700 nm. Total anthocyanin content was calculated and expressed as g cyanindin-3-glucoside equivalent in 100 g extract (g% C-3-GE). Each sample was done in triplicate and the average result was calculated. Product development Extract from ripe fruits of mamao prepared by decoction and evaporation on a water bath (PDE) which promoted the best antioxidant activity and high phenolic and anthocyanin contents was developed as a soluble drinking powder using wet granulation method. The product was determined for physical characteristics, loss on drying, TLC and IR fingerprint and evaluated for antioxidant activity using DPPH scavenging assay compared with ascorbic acid (vitamin C) standard curve. Antioxidant effect of soluble drinking powder from mamao fruit extract was expressed as g vitamin C equivalent per 1 sachet (14 g) of soluble drinking powder. Loss on drying Loss on drying of soluble drinking powder of mamao fruits was investigated using method according to USP 36 [7]. Soluble drinking powder (2 g) was dried in the hot air oven at 105 º C for 3 hours and then every 1 hour until constant weight is obtained and calculated for percent loss on drying. Loss on drying of soluble drinking powder was done in triplicate and the average result was calculated. Thin layer chromatographic (TLC) fingerprint Decoction extract from the ripe fruits of A. ghaesembilla (PDE) was extracted with 0.5 % HCl in methanol (1:10 w/v). The obtained hydrochloric acid methanol fraction was analyzed by thin layer chromatography on TLC pre-coated silica gel 60 GF 254 plate using n-butanol-acetic acid-water (4:1:5) as solvent system. TLC plates were detected under UV 254 and 366 nm and DPPH spray reagent. Infrared spectrometric (IR) fingerprint The infrared spectra of soluble drinking powder of mamao fruits was identified by FTIR (KBr disc) techniques. Statistical analysis All data are reported as means ± standard deviation of triplicates. Least significant difference was used to compare means (p < 0.05). 3. RESULTS Free radical scavenging activity by DPPH scavenging assay Comparing between purple fruit extracts of mamao from various methods of extraction, extract from decoction promoted higher free radical scavenging activity than maceration and squeezing extracts. There is no significant difference in free radical scavenging effect among decoction extracts obtained from various drying methods. The red fruit extract also exhibited high free radical scavenging effect (Table 1). Determination of total phenolic content by Folin-Ciocalteu method As shown in Table 1, RDF exhibited the highest total phenolic content of 2.81 ± 0.88 g% GAE. Total phenolic contents of extracts from the purple fruits of mamao prepared from different methods of extraction ranged from 0.87 ± 1.00 to 2.63 ± 1.02 g% GAE. Determination of total anthocyanin content by ph differential method Squeezing extract from the purple fruits of mamao contained the highest amount of total anthocyanin content of 61.51±0.95 g%c-3-ge (Table 1). The decoction extracts from purple fruits of mamao obtained by freeze drying and evaporation on a water bath significantly contained higher amount of total anthocyanin contents than extract from spray drying. The results suggested that the high temperature during the process of spray drying had a significant decreasing effect to anthocyanin content. From the results, the ripe purple fruit extract prepared from decoction and evaporation on a water bath method (PDE) which exhibited high DPPH scavenging activity, high amount total phenolic and total anthocyanin contents was selected for development of soluble drinking powder. Moreover, this extract contained the high percentage of yield and the extraction and drying procedures were convenient and simple which suitable for upscaling in manufacturing process. www.natpro5.psu.ac.th 280
Table 1. Free radical scavenging activity, total phenolic and total anthocyanin contents of extracts from the fruits of mamao prepared by different extraction and drying methods Sample Yield (%w/w) DPPH assay* Total phenolic* Total anthocyanin* (IC 50, µg/ml) (g% GAE) (g% C-3-GE) PDF 32.56 70.14 ± 3.90 a 1.18 ± 0.44 a 6.03 ± 0.65 a PDS 6.96 62.59 ± 6.63 a 1.68 ± 0.46 a 4.70 ± 0.23 b PDE 28.48 71.26 ± 1.57 a 1.22 ± 0.36 a 7.09 ± 0.24 a RDF 3.38 72.42 ± 3.52 a 2.81 ± 0.88 b 15.79 ± 2.09 c PME 15.76 575.18 ± 17.14 b 0.87 ± 1.00 c 6.21 ± 0.57 a PMM 10.95 3552.23 ± 393.03 c 1.72 ± 1.56 a 6.55 ± 1.04 a PMA 6.91 541.70 ± 51.79 b 1.49 ± 0.86 a 6.56 ± 0.84 a PS 7.06 120.73 ± 5.75 d 2.63 ± 1.02 d 61.51 ± 0.95 d Trolox - 5.49 ± 0.34 e - - PDF = purple fruit decoction freeze drying extract, PDS = purple fruit decoction spray drying extract, PDE = purple fruit decoction water bath evaporating extract, RDF = red fruit decoction freeze drying extract, PME = purple fruit maceration ethanol extract, PMM = purple fruit maceration methanol extract, PMA = purple fruit maceration acetone extract, PS = purple fruit squeezing extract. Different letter in the same column indicated significant differences (p < 0.05). Product development Soluble drinking powder from decoction extract of ripe mamao fruit was developed as a pinkish red powder. In 1 sachet of 14 g preparation contained 3.08 ± 0.81 g%c-3-ge and exhibited free radical scavenging activity equivalent to 0.004 g of vitamin C determined by ph differential method and DPPH scavenging assay, respectively. The soluble drinking powder of mamao fruit exhibited loss on drying of 0.22 ± 0.03%. After applying 1 sachet of soluble drinking powder in 1 glass of water (240 ml), the solution appeared as brownish red solution with sour-sweet flavor. The physical appearances of soluble drinking powder and the drinking solution were shown in Figure 1. A B Figure 1. Soluble drinking powder from decoction extract of mamao fruits (A) and the drinking solution (B) TLC fingerprints Thin layer chromatogram of decoction extract from mamao fruits (PDE) showed specific fingerprints with some dark quenching and fluorescence bands by the detection under UV 254 and 366 nm, respectively. The results suggested the presences of phytochemicals with the chromophores such as conjugated double bonds of phenolics and flavonoids. Moreover, these compounds also showed positive results to DPPH spray reagent appeared as pale yellow bands on purple background suggesting the antioxidant effects (Figure 2). Compound appeared at Rf value of 0.74 was the major band which also promoted antioxidant activity and could be used as an antioxidant marker for quality control of mamao fruit extract in a future. www.natpro5.psu.ac.th 281
Rf = 0.74 A B C Figure 2. TLC fingerprint of decoction extract from mamao fruits (DE) stationary phase: silica gel GF 245 Solvent system: n-butanol-acetic acid-water (4:1:5) Detection: A= UV 254 nm, B= UV 366 nm, C= DPPH spray reagent IR fingerprint Using IR spectroscopic technique (KBr disc), decoction extract from mamao fruits (PDE) promoted the major IR spectra of some functional groups including hydroxyl group (OH), C-H group, keto group (C=O), C=C group, C- O group and fingerprint of benzene ring corresponded to the functional groups of phenolic and flavonoid compounds. The results from IR analysis supported the results from TLC and spectrophotometric assays. The functional groups from IR spectra of soluble drinking powder from mamao fruit extract was described in Table 2 [8]. Table 2. IR spectra of soluble drinking powder from mamao fruit extract Peak Frequency (cm-1) Functional group 1 3403 OH- stretching 2 2970 methyl C-H stretching 3 2901 methylene C-H stretching 4 1732 C=O 5 1622 C=C stretching 6 1418 CH 2 - bending 7 1281 C-H bending 8 1080 C-O stretching 9 600 882 fingerprint of benzene ring Product specification sheet The physical, chemical and biological properties of soluble drinking powder from mamao fruit extract was described as product specification sheet in Table 3 www.natpro5.psu.ac.th 282
Table 3. Product specification sheet of soluble drinking powder from mamao fruit extract Property Description Raw material Ripe purple fruit of mamao (Antidesma ghaesembilla) Method of extraction Decoction Method of drying Evaporation on a water bath Physical characteristic Fined powder Color Pinkish red Odor Odorless Taste of the solution Sour-sweet Weight / sachet (g) 14 How to use 1 sachet soluble in 1 glass of water (240 ml) Total anthocyanin content* of 1 sachet (g% GAE) Not less than 3 (*ph differential method) Antioxidant activity of 1 sachet* Not less than 4 mg ascorbic acid equivalent (*DPPH scavenging assay) Loss on drying (%) Less than 1 TLC fingerprint* (*condition as mentioned above) Specific fingerprint as shown in figure 2 with chromatographic band at Rf = 0.74 which quenching under UV 254 nm and positive to DPPH spray reagent IR fingerprint (KBr disc) Specific IR spectra with peaks at wavenumbers of 3403, 2970, 2901, 1732, 1622, 1418, 1281, 1080, fingerprint around 700-800 corresponded to the functional groups as mentioned in Table 2 4. CONCLUSIONS Extract from the ripe purple fruits of mamao (Antidesma ghaesembilla) prepared by decoction and drying by evaporation on water bath (PDE) was found to promoted high antioxidant activity tested by DPPH scavenging assay. This extract contained high amount of phenolic and anthocyanin contents. Soluble drinking powder from PDE was developed by wet granulation method. The finished product appeared as pinkish red powder that contained 3.08 ± 0.81 g%c-3-ge in 1 sachet (14 g) and exhibited free radical scavenging activity equivalent to 0.004 g of vitamin C. Thin layer chromatographic and infrared spectroscopic fingerprints and loss on drying were conducted to qualitatively control the specification of the product. Investigation for the stability, toxicity and other related biological activities of the soluble drinking powder should be performed in the future. The results of this study could be the guideline for further developments of antioxidant products from mamao fruit extracts in industrial enterprises. ACKNOWLEDGEMENTS Authors would like to express their gratitude to Faculty of Pharmacy, Mahidol University for providing financial support and research facilities. REFERENCES 1. Cross EC. 1987. Oxygen radicals and human disease. Ann. Intern. Med. 107, 526-45. 2. Phargarden.com [homepage on the Internet]. Ubon Ratchathanee. Faculty of Pharmaceutical Sciences, Ubon Ratchathanee University. [updated 2010; cited 2014 Feb 9]. Available from: http://www.phargarden.com/main.php?action=viewpage&pid=95 3. Greenerald.com [homepage on the Internet]. Bangkok. [updated 2013; cited 2014 Feb 9] Available from: http://www.greenerald.com/%e0%b8%a1%e0%b8%b0%e0%b9%80%e0%b8%a1%e0%b9%88%e0%b8 %B2/ 4. Yamasaki K, Hashimoto A, Kokusenya Y, Miyamoto T, Sato T. 1994. Electrochemical method for estimating the antioxidative effects of methanol extracts of crude drugs. Chem Pharm Bull. 42 (8), 1663-1665. www.natpro5.psu.ac.th 283
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